Polypeptide and process for producing the same

ABSTRACT

The invention provides for a polypeptide, which is useful as a biomaterial and free from infection. The polypeptide comprises a peptide unit of formula (1), and optionally one or more units of formulae (2) to (3): (1) [—(OC—(CH 2)   m —CO) p -(Pro-Y-Gly) n -] a ; (2) [—(OC—(CH 2 ) m —CO) q -(Z) r -] b ; and (3) [—HN—R—NH—] c , wherein “m” is 1-18 , “p” and “q” are identical or different, and each is 0 or 1 , “Y” is Pro or Hyp, and “n”1-20 ; “Z” is a peptide chain comprising 1-10 amino acids, “r”1-20 , and “R” is a linear or branched alkylene group; the molar ratio of “a” to “b”[a/b] is 100/0 to 30/70 ; when p=1 and q=0, c=a, when p=0 and q=1, c=b, when p=1 and q=1, c=a+b, and when p=0 and q=0, c=0; and the polypeptide shows a peak of molecular weight in a range from 1×10 4  to 100×10 4  in the molecular weight distribution.

This application is related to and claims priority to Japanese patentapplication No. 53299/2002, filed Feb. 28, 2002.

FIELD OF THE INVENTION

The present invention relates to a novel polypeptide and a process forproducing the same. More specifically, the present invention relates toa novel polypeptide useful as biomaterial which is free from a risk ofan infection by a pathogenic organism (or a causative factor) or anundesirable side effect, and which has a high safety, and a process forproducing the same. Such a biomaterial or biocompatible materialincludes, for example, a medical material such as a carrier or supportfor a tissue engineering, a carrier or support for a regenerativemedical treatment, a tissue binding agent or an antiadhesive material, asuture for a surgical operation, a hemostatic material and a contactlens; a raw material for a pharmaceutical preparation; a raw materialfor a cosmetic preparation; and others.

BACKGROUND OF THE INVENTION

A collagen is a fibrous protein found in all multicellular organisms.The collagen is a main component of skins or bones, and occupies 25% oftotal proteins in mammals. A typical collagen molecular has a rope-likesuperhelical structure, which is referred to as a triple helicalstructure, comprising three collagen polypeptide chains. The collagen isparticularly rich in proline (Pro) and glycine (Gly). These two aminoacid residues are important to form a stable triple helical structure ofthe collagen.

As methods for using a collagen as a biomaterial, there may bementioned, for example, a method of grafting or transplanting an intactor lyophilized skin tissue derived from a pig on a skin area damaged bya burn or scald, a method of removing cellular components from a tissuewith enzyme treatment, and a method of using a collagen which issolubilized by a treatment with an acidic solution or an enzyme toreconstitute a desirable form. A common preparation method and a commonqualitative method are described in Methods Enzymol., Vol. 82, pp. 33 to64, 1982.

There are various suggestions to utilize a collagen. For example,Japanese Patent Application Laid-Open No. 08-027192 (JP-08-027192A)discloses a production process of a collagen derivative for impartingmoisture and smoothness to skin, which comprises esterifying andmodifying an animal tissue containing a collagen with alcohol, andextracting the modified collagen, as well as a cosmetic base materialusing the collagen derivative.

Japanese Patent Application Laid-Open No. 07-097454 (JP-07-097454A)discloses a production process of a water-soluble crosslinked collagenwhich shows a high regeneration rate of a triple helical structure afterthermal denaturation, and the process comprises subjecting awater-soluble collagen to a crosslinking treatment with a bifunctionalalkylene diimidate cross-linker having imide ester groups at both endsof the methylene chain.

Japanese Patent Application Laid-Open No. 08-053548 (JP-08-053548A)discloses a matrix of a collagen and a synthetic polymer (acollagen-synthetic polymer matrix) which has a low immunogenicity and isuseful for preparation of biocompatible implants utilized for variousmedical applications, and a production process of the matrix comprisesreacting a collagen with a first synthetic hydrophilic polymer to form acollagen-synthetic polymer matrix, and further reacting thecollagen-synthetic polymer matrix with a reactant such as a secondsynthetic hydrophilic polymer, a biologically active substance, aglycosaminoglycan and a derivative thereof, a chemical crosslinkingagent, an esterifying agent, an amidating agent, an acylating agent, anamino acid, a polypeptide, or others.

Japanese Patent Application Laid-Open No. 07-278312 (JP-07-278312A)discloses a unit material containing a hydrophilic synthetic polymercovalently bonded to a chemically modified collagen which issubstantially a nonfiberous form at pH 7. The literature discloses thatthe unit material is particularly useful for ophthalmological devicesand optically transparent, and that the unit material has abiocompatibility.

Japanese Patent Application Laid-Open No. 05-000158 (JP-05-000158A)discloses a production process of a collagenic membrane-like substance,which comprises crushing a collagen matrix, centrifuging the crushedmatrix under a high centrifugal field, homogenizing the resultantprecipitate to obtain a paste, casting the paste, and drying the castpaste at a temperature of not higher than 37° C. The literature alsodiscloses that the collagen membrane-like substance has abiocompatibility and a non-inflammatory property, and is useful forrepairing a tissue as an artificial implantation matter.

Japanese Patent Application Laid-Open No. 05-125100 (JP-05-125100A)discloses a soluble fish scale collagen having high-purity and aproduction process thereof, and the process comprises pepsinating anintact or deashed fish scale to a pepsin treatment.

Japanese Patent Application Laid-Open No. 06-228506 (JP-06-228506A)discloses a production process of a dry particulate or powdery solublecollagen, which comprises injecting a collagen solution through a nozzleinto 70 to 90% ethanol medium to form a strand-like or membranousproduct, drying the product, and chopping or grinding the dried product.

Japanese Patent Application Laid-Open No. 08-276003 (JP-08-276003A)discloses use of an unbaked single-crystal hydroxyapatite as a materialfor repairing a biological hard tissue (such as a bone), throughattaching the single crystal to at least part of a low antigeniccollagen fiber.

Japanese Patent Application Laid-Open No. 08-041425 (JP-08-041425A)discloses a method which comprises removing fragments of cells ortissues in a collagen solution and subjecting the residue to an alkalitreatment, for removing prion in a collagen derived from an animal orhuman being, and discloses a collagen obtained by this method.

Moreover, regarding methods for chemical synthesis of collagenanalogues, it has been reported that a soluble polyamide having amolecular weight of 16,000 to 21,000 is obtained by dissolvingp-nitrophenyl ester of Pro-Ser-Gly or p-nitrophenyl ester of Pro-Ala-Glyin dimethylformamide, and adding triethylamine thereto, and allowing tostand the mixture for 24 hours (J. Mol. Biol., Vol. 63, pp. 85 to 99,1972). In this literature, the soluble polyamide is estimated to form atriple helical structure based on the circular dichroism spectra.However there are not referred to properties of the obtained polymer.

It also has been reported that a method for obtaining a polyamide, whichcomprises dissolving a 50-mer peptide containing the sequenceVal-Pro-Gly-Val-Gly derived from elastin in dimethylsulfoxide, adding 2equivalents of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, 1equivalent of 1-hydroxybenzotriazole and 1.6 equivalents ofN-methylmorpholine thereto, allowing to stand the mixture for 14 days,and dialyzing the resultant mixture with a dialysis membrane (molecularweight cut-off: 50,000) (Int. J. Peptide Protein Res., Vol. 46, pp. 453to 463, 1995).

Meanwhile, as described in the above-mentioned Japanese PatentApplication Laid-Open No. 08-041425 (JP-08-041425A), a causativesubstance of sheep tremor or bovine spongiform encephalopathy is aninfectious protein called as prion, and the infectious protein isconsidered as one of causes of human Creutzfeldt-Jakob diseaseinfection. Prion is a protein, and it is indicated that prion is hard todeactivate with a conventional pasteurization or sterilization method,further that prion is infectious over species (Nature Review, Vol. 2,pp. 118 to 126, 2001).

In general, a collagen derived from bovine or pig is frequently used asa raw material for medical kits (devices) or pharmaceuticalpreparations, and cosmetic preparations. Accordingly, there have beenalways existed the risk of an infection (or a transmission) topathogenic organisms or a causative factor such as prion which cannot beremoved by conventional pasteurization or sterilization.

Moreover, since various cell adhesion sites are found in a naturallyoccurring collagen, the naturally occurring collagen cannot exert cellselectivity for any applications. For example, in the case using acollagen as a material for inducing a nerval axon, migration or growthrate of surrounding fibroblast is more than elongation rate of the axonresulting in forming scarring tissue, and the axon cannot be elongated.It is therefore necessary to take a step to cover around the collagenwith a material for protecting migration of fibroblast, or others.

SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to provide a novelpolypeptide which is free from a risk of an infection (or atransmission) by a pathogenic organism (or a causative factor) or anundesirable side effect, and a process for producing the same.

It is another object of the present invention to provide a novelpolypeptide useful as a highly safe biomaterial or biocompatiblematerial, and a process for producing the same.

It is further object of the present invention to provide a process forefficiently producing a polypeptide having above-mentioned propertieswith inhibiting dimerization or cyclization reaction.

The inventors of the present invention made intensive studies to achievethe above objects and finally found that a collagen-like polypeptide canbe produced without cyclization by condensation of a specific peptidecomponent. The present invention was accomplished based on the abovefindings.

That is, the novel polypeptide of the present invention comprisespeptide units represented by the following formulae (1) to (3):[—(OC—(CH₂)_(m)—CO)_(p)—(Pro—Y—Gly)_(n)—]_(a)  (1)[—(OC—(CH₂)_(m)—CO)_(q)—(Z)_(r)—]_(b)  (2)[—HN—R—NH—]_(c)  (3)

wherein “m” denotes an integer of 1 to 18, “p” and “q” are the same ordifferent, each representing 0 or 1, “Y” represents Pro or Hyp, and “n”denotes an integer of 1 to 20; “Z” represents a peptide chain comprising1 to 10 amino acid residue(s), “r” denotes an integer of 1 to 20, and“R” represents a linear or blanched alkylene group; the molar ratio of“a” relative to “b” [a/b] is 100/0 to 30/70;

-   when p=1 and q=0, c=a,-   when p=0 and q=1, c=b,-   when p=1 and q=1, c=a+b, and-   when p=0 and q=0, c=0.

In the polypeptide comprising the units represented by the aboveformulae (1) to (3), usually, “m” denotes an integer of 2 to 12; “n”denotes an integer of 2 to 15; “Z” is a peptide chain comprising atleast an amino acid residue or a peptide residue selected from the groupconsisting of Gly, Sar, Ser, Glu, Asp, Lys, His, Ala, Val, Leu, Arg,Pro, Tyr, and Ile. Moreover, in the above formula, usually, “r” denotesan integer of 1 to 10; and “R” represents a C₂₋₁₂alkylene group.

For example, the polypeptide of the present invention may comprise thefollowing repeating unit (i), (ii) or (iii):

-   (i) a repeating unit comprising the peptide unit [-(Pro—Y—Gly)_(n-])    _(a) (wherein “Y” and “n” have the same meanings as defined above)    and the peptide unit [-(Z)_(r)-]_(b) (wherein “Z” and “r” have the    same meanings as defined above) in a ratio “a/b” of 100/0 to 40/60    (molar ratio); or-   (ii) a repeating unit comprising the peptide unit    [—(OC—(CH₂)_(m)—CO)—(Pro—Y—Gly)_(n)—]_(a) (wherein “m”, “n” and “Y”    have the same meanings as defined above) and the unit    [—HN—R—NH—]_(c) (wherein “R” has the same meaning as defined above)    in a ratio “a/c” of substantial 1/1 (molar ratio); or-   (iii) a repeating unit comprising the peptide unit    [—(OC—(CH₂)_(m)—CO)—(Pro—Y—Gly)_(n)—]a (wherein “m”, “n” and “Y”    have the same meanings as defined above), the peptide unit    [—(OC—(CH₂)_(m)—CO)—(Z)_(r)—]_(b) (wherein “m”, “r” and “Z” have the    same meanings as defined above) and the unit [—HN—R—NH—]_(c)    (wherein “R” has the same meaning as defined above) in a ratio “a/b”    of 100/0 to 40/60 (molar ratio) and in a ratio “(a+b)/c” of    substantial 1/1 (molar ratio).

The polypeptide of the present invention usually shows positive Cottoneffect at a wavelength in range of 220 to 230 nm and negative Cottoneffect at a wavelength in range of 195 to 205 nm in a circular dichroismspectrum. At least part (part or all) of the polypeptide, therefore,forms a triple helical structure. Moreover, the polypeptide of thepresent invention is capable of forming a collagenous tissue (collagentissue or collagen-like tissue). The polypeptide of the presentinvention may show a peak corresponding to the molecular weight in arange from 5×10³ to 100×10⁴.

According to the present invention, the polypeptide may be produced byreacting a peptide component (A) represented by the following formula(1a) with a peptide component (B) represented by the following formula(2a):X—(Pro—Y—Gly)_(n)—OH  (1a)

-   -   wherein “X” represents H or the group HOOC—(CH₂)_(m)—CO— (“m”        denotes an integer of 1 to 18), “Y” represents Pro or Hyp, and        “n” denotes an integer of 1 to 20,        X—(Z)_(r)—OH  (2a)    -   wherein “X” represents H or the group HOOC—(CH₂)_(m)—CO— (“m”        denotes an integer of 1 to 18), “Z” represents a peptide chain        comprising 1 to 10 amino acid residue(s), and “r” denotes an        integer of 1 to 20.

In the reaction, in the case where “X” represents H in the formula (1a)and/or (2a), the reaction is conducted without the compound (C)represented by the following formula (3a). In the case where “X”represents the group HOOC—(CH₂)_(m)—CO— (“m” has the same meaning asdefined above) in the formula (1a) and/or (2a), the reaction isconducted with the compound (C) represented by the following formula(3a):H₂N—R—NH₂  (3a)

-   -   wherein “R” represents a linear or branched alkylene group.

The ratio of the peptide component (A) relative to the peptide component(B) [the former (A)/the latter (B)] is, for example, about 100/0 to30/70 (mol %), and the amount of the compound (C) is, for example,substantially 1 mol relative to 1 mol of the total molar amount of thepeptide component (A) and/or the peptide component (B).

The reaction may be usually carried out by condensing each component (1)to (3) in a solvent (water and/or an organic solvent) in the presence ofat least a dehydrating and condensing agent (e.g., a carbodiimide-seriescondensing agent, a fluorophosphate-series condensing agent, and adiphenylphosphorylazide). Moreover, the reaction may be carried out inthe presence of the dehydrating and condensing agent and a condensingauxiliary (or a dehydrating auxiliary) [for example, anN-hydroxypolycarboxylic imide, an N-hydroxytriazole (e.g., anN-hydroxybenzotriazole such as 1-hydroxybenzotriazole), a triazine,ethyl ester of 2-hydroxyimino-2-cyanoacetic acid]. In the case where thereaction is conducted in a water-free solvent system, the proportion ofthe dehydrating and condensing agent may be about 0.7 to 5 mol relativeto 1 mol of the total molar amount of the reaction components (1a), (2a)and (3a). In the case where the reaction is conducted in awater-containing solvent system, the proportion of the dehydrating andcondensing agent may be about 2 to 500 mol relative to 1 mol of thetotal molar amount of the reaction components (1a) (2a) and (3a). Theamount of the condensing auxiliary may be about 0.5 to 5 mol relative tothe total molar amount of the above reaction components (1a), (2a) and(3a).

The present invention also includes a method for applying thepolypeptide to a tissue of a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scanning electron micrograph showing a film obtained inExample 3.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, amino acid residues are abbreviated to thefollowing condensation codes.

-   Ala: L-alanine residue-   Arg: L-arginine residue-   Asn: L-asparagine residue-   Asp: L-aspartic acid residue-   Cys: L-cysteine residue-   Gln: L-glutamine residue-   Glu: L-glutamic acid residue-   Gly: glycin residue-   His: L-histidine residue-   Hyp: L-hydroxyproline residue-   Ile: L-isoleucine residue-   Leu: L-leucine residue-   Lys: L-lysine residue-   Met: L-methionine residue-   Phe: L-phenylalanine residue-   Pro: L-proline residue-   Sar: sarcosine residue-   Ser: L-serine residue-   Thr: L-threonine residue-   Trp: L-tryptophan residue-   Tyr: L-tyrosine residue-   Val: L-valine residue

Moreover, in this specification, amino acid sequences of peptide chainsare represented in accordance with conventional expression that, in anamino acid residue, N-terminus and C-terminus are drawn at the left andthe right, respectively.

It is essential that a peptide unit (1)[—(OC—(CH₂)_(m)—CO)_(p)(Pro—Y—Gly)_(n)—] constituting the novelpolypeptide of the present invention contains a repeating sequencePro—Y—Gly. When the repeating number of the sequence Pro—Y—Gly issmaller, the polypeptide is deteriorated in stability of the triplehelical structure. When the repeating number is too large, it isdifficult to synthesize the peptide. The repeating number “n” is,therefore, about 1 to 20, preferably about 2 to 15 (e.g., about 3 to15), and more preferably about 5 to 15.

In the above formula (1), “Y” may be either Pro or Hyp. From theviewpoint of stability of the triple helical structure, the preferred“Y” is Hyp. Incidentally, Hyp is usually 4Hyp (e.g.,trans-4-hydroxy-L-proline) residue.

Further, the number “m” representing the repeating number of a methylenechain (CH₂) needs only to be selected from a range that the polypeptideis adversely affected physical and biological properties. The number “m”is usually about 1 to 18, preferably about 2 to 12, and more preferablyabout 2 to 10 (particularly about 2 to 6). The number “p” is 0 or 1.

In the above-mentioned peptide unit (2) [—(OC—(CH₂)—CO)—(Z)_(r)—], “Z”represents a peptide chain which has an arbitrary sequence comprising 1to 10 amino acid residue(s). “Z” may be any sequence as far as theresulting polypeptide is not deteriorated in physical and biologicalproperties. In order that the polypeptide shows useful physical andbiological properties, for example, the peptide chain “Z” usuallycomprises at least one amino acid or peptide residue selected from thegroup consisting of Gly, Sar, Ser, Glu, Asp, Lys, His, Ala, Val, Leu,Arg, Pro, Tyr and Ile. In particular, the peptide chain “Z” practicallycomprises at least one amino acid or peptide residue selected from thegroup consisting of Gly, Ser, Glu, Asp, Lys, Arg and Pro. The peptidechain “Z” preferably comprises the amino acid residue or the sequencesuch as Gly, Sar, Ser, Glu, Asp, Lys, Arg-Gly-Asp, Tyr-Ile-Gly-Ser-Arg,Ile-Lys-Val-Ala-Val, Val-Pro-Gly-Val-Gly, Asp-Gly-Glu-Ala,Gly-Ile-Ala-Gly, His-Ala-Val, Glu-Arg-Leu-Glu, Lys-Asp-Pro-Lys-Arg-Leu,or Arg-Ser-Arg-Lys.

The number “r” representing the repeating number of the peptide chain“Z” needs only to be selected from a range that the resultingpolypeptide demonstrates physical and biological properties. When therepeating number “r” is too large, it is difficult to synthesize thepeptide, and the physical properties of the resulting polypeptide is aptto vary. The repeating number “r” is, therefore, usually about 1 to 20,preferably about 1 to 10, and more preferably about 1 to 5.

The number “m” representing the repeating number of the methylene chain(CH₂) is about 1 to 18, preferably about 2 to 12, and more preferablyabout 2 to 10 (particularly about 2 to 6) as the same manner as in theabove formula (1). The number “q” is 0 or 1.

In the formulae (1) and (2), in the case where at least one of “p” and“q” is 0, the polypeptide contains the unit [—HN—R—NH—] represented bythe formula (3). In the unit represented by the formula (3), a linear orblanched alkylene group represented by “R” needs only be any group asfar as the polypeptide is not deteriorated in physical and biologicalproperties. For example, as the group “R”, there may be mentioned aC₁₋₁₈alkylene group such as methylene, ethylene, propylene,trimethylene, and tetramethylene groups. The alkylene group “R” may be alinear methylene chain (CH₂)_(s) (“s” represents an integer of 1 to 18).The group “R” is preferably a C₂₋₁₂alkylene group, more preferably aC₂₋₁₀alkylene group, in particular a C₂₋₆alkylene group.

The ratio [“a/b”] of the peptide unit represented by the formula (1)relative to the peptide unit represented by the formula (2) is about100/0 to 30/70 (molar ratio), preferably about 100/0 to 40/60 (molarratio), and more preferably about 100/0 to 50/50 (molar ratio).

Further, the ratio of the unit represented by the formula (3) may beselected depending on the value “p” of the formula (1) and the value “q”of the formula (2). When “p”=1 and “q”=0, “c”=a (=1). When “p”=0 and“q”=1, “c”=b (=1). Moreover, when “p”=1 and “q”=1, “c”=a+b (=2). When“p”=0 and “q”=0, “c”=0.

That is, the polypeptide of the present invention includes the followingpolypeptides:

-   (a) a polypeptide comprising a repeating unit of the peptide unit    [—(Pro—Y—Gly)_(n)—]_(a) (“p” is 0 in the formula (1));-   (b) a polypeptide which comprises a repeating unit containing the    peptide unit [—(Pro—Y—Gly)_(n)—]_(a) (“p” is 0 in the formula (1))    and the peptide unit [—(Z)_(r)—]_(b) (“q” is 0 in the formula (2))    in a ratio of “a/b” (mol %);-   (c) a polypeptide which comprises a repeating unit containing the    peptide unit [—(OC—(CH₂)_(m)—CO)—(Pro—Y—Gly)_(n)—]_(a) (“p” is 1 in    the formula (1)) and the unit [—HN—R—NH—]_(c) represented by the    formula (3) in a ratio of “a/c” (mol %); and-   (d) a polypeptide which comprises a repeating unit containing the    peptide unit [—(OC—(CH₂)_(m)—CO)—(Pro—Y—Gly)_(n)—]_(a) (“p” is 1 in    the formula (1)), the peptide unit [—(OC—(CH₂)_(m)—CO)—(Z)_(r)—]_(b)    (“q” is 1 in the formula (2)) and the unit [—HN—R—NH—]_(c)    represented by the formula (3) in a ratio of “a/b/c” (mol %).

Such a polypeptide forms a linear polypeptide without forming a ringsuch as a six-membered ring by cyclization, and is soluble in a solvent(a hydrophilic solvent, e.g., water, an alcohol such as ethanol andpropanol, a ketone such as acetone, a cyclic ether such as dioxane andtetrahydrofuran, a sulfoxide such as dimethyl sulfoxide, or a mixedsolvent thereof). For example, the polypeptide shows a peakcorresponding to the molecular weight in a range from about 5×10³ to100×10⁴, and preferably from about 1×10⁴ to 10×10⁴ in terms of aglobular protein by means of an aqueous GPC (gel permeationchromatography).

Further, the novel polypeptide of the present invention is capable offorming (formable) a collagenous tissue (collagen tissue orcollagen-like tissue). That is, the polypeptide of the present inventionshows positive Cotton effect at a wavelength in range of 220 to 230 nmand negative Cotton effect at a wavelength in range of 195 to 205 nm incircular dichroism spectra. At least one part (that is, part or all) ofthe polypeptide of the present invention is, accordingly, capable offorming a triple helical structure, and the polypeptide of the presentinvention forms a collagenous (collagen-like) polypeptide. Incidentally,Cotton effect means a phenomenon caused by difference between anabsorption coefficient relative to a right circularly polarized lightand that relative to a left at a specific wavelength in an opticalrotatory substance.

The novel polypeptide of the present invention can be obtained bysubjecting the above-mentioned components (1a) to (3a) to a condensationreaction. The peptide chain of the present invention can be synthesizedin accordance with a conventional peptide synthesis method. Peptidesmay, for example, be prepared based on a solid-phase synthesis method ora liquid-phase synthesis method, and the solid-phase synthesis method isoperationally convenient [for example, see “Zoku Seikagaku Jikken Kouza2, Tanpakushitsu no Kagaku (Supplemental Handbook of BiochemicalExperiments, Chemistry of Protein) (the second volume)” edited by TheJapanese Biochemical Society (issued by Tokyo Kagaku Dozin Co., Ltd.,May 20, 1987), pp. 641 to 694]. For the peptide synthesis, aconventional manner may be utilized, and the manner includes, forexample, a coupling method using a condensing agent, an activeesterification method (e.g., a phenyl ester such as p-nitrophenyl ester(ONp) and pentafluorophenyl ester (Opfp), an N-hydroxydicarboxylic imideester such as N-hydroxysuccinimide ester (ONSu), and1-hydroxybenzotriazole ester (Obt)), a mixed acid anhydride method, anazide method, and others. In the preferred manner, at least a condensingagent (preferably a condensing agent as mentioned below, in particular acombination of a condensing agent as mentioned below with a condensingauxiliary as mentioned below) may be practically used.

Furthermore, in the peptide synthesis, protection of an amino group, acarboxyl group, and other functional group (e.g., a guanidino group, animidazolyl group, a mercapto group, a hydroxyl group, an ω-carboxylgroup) with a protective group, and elimination or removal of theprotective group with a catalytic reduction or a strong acid treatment(e.g., anhydrous hydrogen fluoride, trifluoromethanesulfonic acid,trifluoroacetic acid) are repeatedly conducted depending on a species ofamino acids or peptide fragments. For example, as a protective group foran amino group, there may be utilized benzyloxycarbonyl group (Z),p-methoxybenzyloxycarbonyl group (Z(OMe)), 9-fluorenylmethoxycarbonylgroup (Fmoc), t-butoxycarbonyl group (Boc), 3-nitro-2-pyridinesulfenylgroup (Npys), and the other groups. As a protective group for a carboxylgroup, there may be utilized benzyloxy group (OBzl), phenacyloxy group(OPac), t-butoxy group (OBu), methoxy group (OMe), ethoxy group (OEt),and the other groups. Incidentally, an automatic synthesis apparatus maybe utilized for the peptide synthesis.

More specifically, the preparation of the peptide chain of the presentinvention with the solid-phase synthesis method may be carried out inaccordance with a conventional manner. As a solid-phase resin (or acarrier), there may be utilized a polymer insoluble to a reactionsolvent, for example, a styrene-divinylbenzene copolymer (such as achloromethylated resin, a hydroxymethyl resin, ahydroxymethylphenylacetamidemethyl resin, a 4-methylbenzhydrylamineresin).

In the solid-phase synthesis method, a peptide can be usually producedby the following steps: a step forming a peptide chain corresponding toan objective peptide, and a step comprising (iv) detaching the peptidechain from the polymer (resin) and eliminating the protective group(s)from the protected functional group(s) to obtain the objective peptide,and purifying the resulting peptide. The peptide chain-forming stepcomprises (i) bonding an amino acid or peptide fragment to the abovepolymer (resin) from C-terminal to N-terminal of the objective peptide,in which the amino acid or peptide fragment has a free α-COOH group anda functional group(s) (e.g., at least an α-amino group of theN-terminal) protected with a protective group(s), (ii) eliminating theprotective group of an a-amino group forming a peptide bond among thebonded amino acid or peptide fragment, and (iii) sequentially repeatingthe above bonding operation and the eliminating operation to elongatethe peptide chain for the formation of the object peptide. In theoperation (i) for bonding the amino acid or peptide fragment, an aminoacid which is corresponding to the C-terminal of the peptide chain andhas a free α-COOH group, and in which at least the N-terminal isprotected with a protective group (for example, a Fmoc-amino acid, aBoc-amino acid) is used. Incidentally, from the viewpoint of inhibitinga side reaction, detachment of the peptide chain from the polymer ispreferably carried out concurrently with elimination of the protectivegroup with the use of trifluoroacetic acid. Moreover, the resultingpeptide may be purified by utilizing a separation and purification means(e.g., a reversed phase liquid chromatography, and a gel-permeationchromatography).

In the present invention, a polypeptide is prepared by condensing atleast a peptide component (A) represented by the following formula (1a):X—(Pro—Y—Gly)_(n)—OH  (1a)

wherein “X” represents H or HOOC—(CH₂)_(m)—CO— (“m” has the same meaningas defined above), “Y” and “n” have the same meanings as defined above.

The polypeptide of the present invention may be prepared byco-condensing the peptide component (A) represented by the above formula(1a) with a peptide component (B) represented by the following formula(2a):X—(Z)_(r)—OH  (2a)

wherein X represents H or HOOC—(CH₂)_(m)—CO— (“m” has the same meaningas defined above), “Z” and “r” have the same meanings as defined above.

Incidentally, as the compound in which the above group “X” isHOOC—(CH₂)_(m)—CO—, there may be mentioned, for example, a C₃₋₂₀aliphatic dicarboxylic acid such as malonic acid, succinic acid,glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid andsebacic acid, or an acid anhydride thereof. These compounds may be usedsingly or in combination. These compounds may be also subjected to areaction according to a conventional amide bond-forming method (forexample, a reaction using a tertiary amine described later as acatalyst) or the above-mentioned peptide synthesis method to obtain thecompounds represented by the above formulae (1a) and (2a).

The ratio of the peptide component (A) relative to the peptide component(B) [the former (A)/the latter (B)] is, for example, about 100/0 to30/70 (mol %), preferably about 100/0 to 40/60 (mol %), and morepreferably about 100/0 to 50/50 (mol %).

Further, in the above formulae (1a) and/or (2a), if the group “X” isHOOC—(CH₂)_(m)—CO— (“m” has the same meaning as defined above), at leastone of the peptide component (A) and the peptide component (B) issubjected to a co-condensation reaction with the compound (C)represented by the following formula (3a) for forming an amide group. Ifthe group “X” is H, it is unnecessary to use the compound (C).H₂N—R—NH₂  (3a)

“R” has the same meaning defined above.

As the compound represented by the above formula (3a), there may beexemplified a diamine corresponding to the above formula (3), e.g., aC₁₋ ₁₈alkylenediamine such as ethylenediamine, trimethylenediamine,propylenediamine, tetramethylenediamine and hexamethylenediamine, apolyalkylenepolyamine such as diethylenetriamine andhexamethylenetetramine, and others. These compounds may be used singlyor in combination.

The amount of the diamine compound (C), for example, may besubstantially 1 mol (for example, about 0.95 to 1.05 mol) relative to 1mol of a total molar amount of the peptide component (A) and/or thepeptide component (B). Incidentally, in the case where the group “X” isHOOC—(CH₂)_(m)—CO—(“m” has the same meaning as defined above) in eitherthe peptide component (A) or (B), the amount of the diamine compound (C)may be substantially 1 mol (for example, about 0.95 to 1.05 mol)relative to 1 mol of the peptide component having such group.

The reaction of these components (1a), (2a) and (3a) is usually carriedout in a solvent. The solvent may be capable of dissolving or suspending(partly or wholly dissolving) the peptide components and the compound,and there may be usually employed water and/or an organic solvent. Asthe solvent, there may be mentioned, for example, water, an amide (e.g.,dimethylformamide, dimethylacetamide, hexamethylphosphoramide), asulfoxide (e.g., dimethylsulfoxide), a nitrogen-containing cycliccompound (e.g., N-methylpyrrolidone, pyridine), a nitrile (e.g.,acetonitrile), an ether (e.g., dioxane, tetrahydrofuran), an alcohol(e.g., methyl alcohol, ethyl alcohol, propyl alcohol), and a mixedsolvent thereof. Among these solvents, water, dimethylformamide, ordimethylsulfoxide is practically used.

The reaction of these components (1a), (2a) and (3a) may be usuallycarried out in the presence of at least a dehydrating agent (adehydrating and condensing agent). The reaction with these components inthe presence of a dehydrating and condensing agent and a condensingauxiliary (synergist) produces smoothly a polypeptide with inhibitingdimerization or cyclization.

The dehydrating and condensing agent is not particularly limited as faras the agent can conduct dehydration and condensation efficiently in theabove-mentioned solvent. For example, the dehydrating and condensingagent (the dehydrating agent) includes a carbodiimide-series condensingagent [e.g., diisopropylcarbodiimide (DIPC),1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC=WSCI),1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (WSCI HCl),dicyclohexylcarbodiimide (DCC)], a fluorophosphate-series condensingagent [e.g., O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate, O-benzotriazole-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate, benzotriazole-1-yl-oxy-tris-pyrrolidinophosphoniumhexafluorophosphate, a salt ofbenzotriazole-1-yl-tris(dimethylamino)phosphoniumhexafluorophosphide(BOP)], diphenylphosphorylazide (DPPA), and others. The dehydrating andcondensing agent(s) may be used singly, or used as a mixture incombination thereof. The dehydrating and condensing agent is preferablya carbodiimide-series condensing agent [e.g.,1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide,1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride].

The condensing auxiliary is not particularly limited as the condensingauxiliary can facilitate the reaction of the condensing agent. Forexample, there may be mentioned an N-hydroxypolycarboxylic imide [e.g.,an N-hydroxydicarboxylic imide such as N-hydroxysuccinic imide (HONSu)and N-hydroxy-5-norbornene-2,3-dicarboxylic imide (HONB)]; anN-hydroxytriazole [e.g., an N-hydroxybenzotriazole such as1-hydroxybenzotriazole (HOBt)]; a triazine such as3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HOObt); ethyl ester of2-hydroxyimino-2-cyanoacetic acid; and others. The condensing auxiliarymay be also used singly or in combination. The condensing auxiliary ispreferably an N-hydroxydicarboxylic imide [e.g., HONSu], anN-hydroxybenzotriazole or an N-hydroxybenzotriazine [e.g., HOBt].

The dehydrating and condensing agent may be suitably used in combinationwith the condensing auxiliary. As a combination of the dehydrating andcondensing agent with the condensing auxiliary, there may be mentioned,for example, DCC-HONSu (HOBt or HOObt), WSCI-HONSU (HOBt or HOObt), andother combinations.

The amount of the dehydrating and condensing agent is, in a water-freesolvent system, usually about 0.7 to 5 mol, preferably about 0.8 to 2.5mol, and more preferably about 0.9 to 2.3 mol (e.g., about 1 to 2 mol)relative to 1 mol of the total molar amount of the reaction components(1a), (2a) and (3a). In a water-containing solvent system, since thedehydrating and condensing agent may be deactivated by water, the amountof the dehydrating and condensing agent is usually about 2 to 500 mol(e.g., about 2 to 50 mol), preferably about 5 to 250 mol (e.g., about 5to 25 mol), and more preferably about 10 to 125 mol (e.g., about 10 to20 mol) relative to 1 mol of a total molar amount of the reactioncomponents (1a), (1b) and (1c). The amount of the condensing auxiliaryis, for example, about 0.5 to 5 mol, preferably about 0.7 to 2 mol, andmore preferably about 0.8 to 1.5 mol relative to 1 mol of a total molaramount of the reaction components (1a), (1b) and (1c) irrespective of akind or species of solvent.

In the condensation reaction of the present invention, the pH of thereaction system may be adjusted, or a base being inert for the reactionmay be added to the system. The pH may be usually adjusted with aninorganic base [e.g., sodium hydroxide, potassium hydroxide, sodiumcarbonate, sodium hydrogen carbonate], an organic base, an inorganicacid [e.g., hydrochloric acid], or an organic acid. The pH of thereaction mixture is usually adjusted to approximately neutral pH(pH=about 6 to 8). As the base being inert for the reaction, there maybe exemplified a tertiary amine (e.g., a trialkylamine such astrimethylamine, triethylamine and diisopropylethylamine, a heterocyclictertiary amine such as N-methylmorpholine and pyridine), and others. Theamount of the base may be usually selected within a range from one totwo times as much as the total molar amount of amino groups in thepeptide components and the compound.

In the present invention, both the formation of the peptide chainrepresented by the above formula X—(Pro—Y—Gly)_(n)—OH, and the formationof a triple helical structure in the resulting polypeptide can beusually proved by measuring circular dichroism spectra or gel-permeationchromatography for a solution of the peptide chain or the polypeptide.In particular, regarding circular dichroism spectra, it has beenreported that a naturally-occurring collagen and peptide chain forming atriple helical structure distinctively shows positive Cotton effect at awavelength in range of 220 to 230 nm and negative Cotton effect at awavelength in range of 195 to 205 nm (J. Mol. Biol., Vol. 63 pp. 85 to99, 1972). Moreover, in accordance with gel-permeation chromatography,since a peak corresponding to a molecular weight of a trimer forming atriple helical structure is observed away from a peak corresponding tothat of a monomer, the proportion of the peptide forming the triplehelical structure can be determined.

The polypeptide of the present invention is capable of forming acollagenous tissue, free from a risk of an infection of a pathogenicorganism or a transmission of a causative factor (e.g., a proteinconverted into a pathological protein, such as abnormal prion) or anundesirable side effect, and has high safety. Moreover, the polypeptideis excellent in cytophilic property and biocompatibility. Thepolypeptide is, therefore, useful as a biomaterial or a biocompatiblematerial, for example, an artificial collagen. The polypeptide of thepresent invention may be applied to a tissue (e.g., an epidermal tissueand a dermal tissue) of a subject. The subject includes human beings,and nonhumans (such as monkeys, sheep, bovines, horses, dogs, cats,rabbits, rats, and mice). Moreover, the polypeptide of the presentinvention may be used for inhibiting or preventing an infection (or atransmission) originated from (caused by) a polypeptide (e.g., aninfection or a transmission of a pathogenic organism or a causativefactor present in a polypeptide). Accordingly, the polypeptide of thepresent invention may be effectively utilized in a damaged area [forexample, a diseased area or an injured area (e.g., an area damaged by ascratch and a burn or scald)] or a dissected (or an incised) area [forexample, a dissected area such as a surgical cut]. For example, thepolypeptide is utilized as a medical material such as a carrier for atissue engineering, a carrier for a regenerative medical treatment(e.g., an artificial skin), a tissue binding agent or an antiadhesivematerial, a suture for a surgical operation, a hemostatic material and acontact lens; a raw material (or base material) for a pharmaceuticalpreparation; a raw material (or base material) for a cosmeticpreparation; a food additive; and others.

The polypeptide of the present invention can be shaped or molded by aconventional manner depending on various applications. The form to beused of the polypeptide may be a liquid form (e.g., a solution, asuspension), a particulate form, a two-dimensional form (e.g., a film, asheet), or a three-dimensional form. For example, a sheet or film of thepolypeptide may be obtained by casting a solution or suspension of thepolypeptide on a releasable substrate (support) (e.g., a sheet made froma fluorine-containing resin (polytetrafluoroethylene)) and drying thecast substrate. Moreover, a fibrous substance is obtained by extruding asolution or suspension of the polypeptide through a nozzle in a solutioncontaining a salt of high concentration or in a solvent incapable ofdissolving the polypeptide. Further, a gelatinous substance may beobtained by allowing to stand an aqueous solution or suspension of thepolypeptide, or if necessary, with adding a polyvalent crosslinkablereagent (e.g., glutaraldehyde) thereto. Further, a sponge-like poroussubstance may be obtained by lyophilizing the resultant gelatinoussubstance. Furthermore, a porous substance can be also obtained bystirring the aqueous solution or suspension of the polypeptide to foam,and drying.

Further, the polypeptide of the present invention may be used as acoating agent. For example, a surface of a substrate may be coated withthe polypeptide of the present invention by coating or spraying with thesolution or suspension of the polypeptide and drying the coated orsprayed layer. The substrate may be a shaped article made of variousmaterials such as a metal, a ceramic, a plastic, a natural polymer, orthe like. The form of the shaped article may be a two-dimensionalstructure or a three-dimensional structure, e.g., a particulate form, alinear or fibrous form, a film or sheet form. Further, the polypeptidemay be held or supported by impregnating, into the solution orsuspension of the polypeptide, a porous substance (e.g., a particulateporous substance, a two-dimensional porous substance such as a papermade from a cellulose fiber (cellulosic paper), a nonwoven or wovenfabric, a three-dimensional porous substance such as a cylindricalsubstance).

In the case using the polypeptide of the present invention for a medicalpurpose, the polypeptide is preferably sterilized or pasteurized. Asterilization or pasteurization method includes various methods, forexample, pasteurization with steam such as a heated and damp steam,pasteurization with a gamma ray, pasteurization with ethylene oxide gas,sterilization with a pharmaceutical preparation, sterilization with anultraviolet ray, and others. Among these methods, pasteurization with agamma ray and pasteurization with ethylene oxide gas are preferred fromthe viewpoint of pasteurization efficiency and low (or light) adverseeffects on a material to be used.

The novel polypeptide of the present invention has no fear of a risk ofan infection by a pathogenic organism (or a causative factor) or anundesirable side effect, with a high safety, and a high cytophilicproperty. Further, the polypeptide can be produced by a simple operationsuch as a condensation reaction without inhibition of dimerization orcyclization.

EXAMPLES

The following examples are intended to describe this invention infurther detail and should by no means be interpreted as defining thescope of the invention.

Example 1

A peptide represented by the formula H—(Pro-Pro-Gly)₁₀—OH (Sequence IDNo. 1) (manufactured by Peptide Institute, Inc.) was dissolved in watercontaining 50% methyl alcohol at a concentration of 0.25 mg/mL, and acircular dichroism spectrum thereof was measured with a spectrometer(J-725, manufactured by JASCO Corp., light path: 1 mm). The resultshowed that positive Cotton effect was observed at a wavelength of 227nm and negative Cotton effect at a wavelength of 197 nm were observed,and confirmed that the peptide formed a triple helical structure.Moreover, with respect to the same solution, the molecular weight wasmeasured by gel-permeation chromatography (AKTA purifier system,manufactured by Amarsham Bioscience K.K., column: Superdex Peptide PE7.5/300, flowrate: 0.25 mL/min., eluent: 10 mM phosphate buffer (pH 7.4)containing 150 mM NaCl). The result confirmed two peaks: one peakshowing a molecular weight of about 9000 corresponding to a triplehelical structure, and another peak showing a molecular weight of about3000 corresponding to a monomer. The molecular weight was calculatedwith a Gel Filtration LMW Calibration Kit (manufactured by AmarshamBioscience K.K.), human insulin (manufactured by Sigma Corporation) andglycine (manufactured by Wako Pure Chemical Industries, Ltd.) asreference materials. The proportion of the peptide forming the triplehelical structure was about 50% on the basis of two peak areas.Incidentally, in the case using a solution dissolving the peptideH—(Pro-Pro-Gly)₁₀—OH in water at a concentration of 0.25 mg/mL, weakpositive Cotton effect at a wavelength of 227 nm and negative Cottoneffect at a wavelength of 198 nm were observed. However, a peakcorresponding to a trimer was inappreciably found in gel-permeationchromatography, and the proportion of the peptide forming the triplehelical structure was almost 0%.

Five (5) mg (0.002 mmol) of the peptide H—(Pro-Pro-Gly)₁₀—OH wassuspended in 2 mL dimethyl sulfoxide, and the mixture was stirred at aroom temperature. To the mixture were added 0.31 mg (0.0024 mmol) ofdiisopropylethylamine, 0.32 mg (0.0024 mmol) of 1-hydroxybenzotriazole,and 0.46 mg (0.0024 mmol) of1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, and theresultant mixture was further stirred for 7 days at a room temperature.

The reaction mixture (solution) was diluted 20-fold with water, and thediluted solution was subjected to a gel-permeation chromatography (AKTApurifier system, manufactured by Amarsham Bioscience K.K., column:Superdex 200 HR10/30, flowrate: 0.5 mL/min., eluent: 10 mM phosphatebuffer (pH 7.4) containing 150 mM NaCl). The result confirmed apolypeptide peak in a molecular weight range of 40000 to 200000. Themolecular weight was calculated with a Gel Filtration LMW CalibrationKit and a Gel Filtration HMW Calibration Kit (manufactured by AmarshamBioscience K.K.) as reference materials.

The resulting reaction mixture was diluted 5-fold with water, and thediluted solution was dialyzed against water for 3 days to remove thereagent(s) such as the condensing agent and the unreacted monomer(s).The circular dichroism spectrum was measured in the obtainedpolypeptide, and the result showed that positive Cotton effect wasobserved at a wavelength of 227 nm and negative Cotton effect at awavelength of 199 nm, and confirmed that the polypeptide formed a triplehelical structure.

Example 2

A peptide chain represented by the formula H—(Pro-Pro-Gly)₅—OH (SequenceID No. 2) was synthesized by a solid-phase synthesis with an automaticpeptide synthesis machine. That is, with the use of 0.1 mmol of aparticulate resin [HMP glycine, manufactured by Applied Biosystems (US)]which comprises a styrene-divinylbenzene copolymer containing4-(Na-9-(fluorenylmethoxycarbonyl)-glycine)-oxymethyl-phenoxy-methylgroup in a proportion of 0.65 mmol/g (resin) [component molar ratio ofstyrene relative to divinylbenzene: 99/1], the carboxyl terminal of oneamino acid was sequentially linked (or bound) to the amino terminal ofthe other amino acid so as to obtain the object peptide. In this linkreaction, 1 mmol of Nα-9-(fluorenylmethoxycarbonyl)-L-proline[Fmoc-proline] and 1 mmol of Nα-9-(fluorenylmethoxycarbonyl)-glycine[Fmoc-glycine] (manufactured by Applied Biosystems (US)) were used asamino acids in each linking step.

The peptide resin obtained in the foregoing manner was suspended in 10mL of dimethylformamide, and 50 mg (0.5 mmol) of succinic anhydride and13 mg (0.1 mmol) of diisopropylethylamine were added thereto. Themixture was subjected to a reaction at a room temperature for 12 hours.Thereafter, the reaction mixture was washed with methyl alcohol anddichloromethane alternately, and dried under a reduced pressure.

The peptide resin obtained by the above-mentioned manner was subjectedto a treatment with 10 mL of trifluoroacetic acid containing 5% waterfor 3 hours. The resulting solution was added to diethyl ether to form aprecipitate, and the precipitate was further washed with diethyl etherseveral times to deprotect the peptide and to eliminate the peptide fromthe resin. The resulting crude product was purified by a PD10 column(manufactured by Amarsham Bioscience K.K.) to give a peptide. Thepurified peptide obtained in the foregoing manner was run through “AKTAexplorer10XT” manufactured by Amarsham Bioscience K.K. [column:“Nova-Pak C18”, manufactured by Millipore Corporation, 3.9 mmφ×150 mm,mobile phase: a mixed solvent of acetonitrile and water containing 0.05vol. % of trifluoroacetic acid (concentration of acetonitrile waslinearly increased from 5 to 50 vol. % for 30 minutes), flow rate: 1.0mL/min.], and a single peak was shown at a retention time of 14.5minutes. The molecular weight of the purified peptide was determined as1375 based on FAB method mass spectrum (theoretical value: 1374.52).

The compound HOOC—(CH₂)₂—CO—(Pro-Pro-Gly)₅—OH obtained by theabove-mentioned manner was dissolved in water or water containing 50% ofmethyl alcohol to prepare a solution having a concentration of 0.25mg/mL, and a circular dichroism spectrum thereof was measured with aspectrometer (J-725, manufactured by JASCO Corp., light path: 1 mm). Theresults in both solutions showed that no positive Cotton effect wasobserved at a wavelength in range of 220 to 230 nm and only negativeCotton effect was observed at a wavelength in range of 200 to 202 nm,and confirmed that the peptide did not form a triple helical structure.Moreover, with respect to the same solution, the molecular weight wasmeasured by gel-permeation chromatography (AKTA purifier system,manufactured by Amarsham Bioscience K.K., column: Superdex Peptide PE7.5/300, flowrate: 0.25 mL/min., eluent: 10 mM phosphate buffer (pH 7.4)containing 150 mM NaCl). The result confirmed one peak showing amolecular weight of not more than 2000 corresponding to a monomer. Themolecular weight was calculated with a Gel Filtration LMW CalibrationKit (manufactured by Amarsham Bioscience K.K.), human insulin(manufactured by Sigma Corporation) and glycine (manufactured by WakoPure Chemical Industries, Ltd.) as reference materials. The proportionof the peptide forming the triple helical structure was 0% on the basisof the peak area.

The compound HOOC—(CH₂)₂—CO—(Pro-Pro-Gly)₅—OH (1.4 mg (0.001 mmol)) andethylenediamine (0.06 mg (0.001 mmol)) were suspended in 0.05 mL water,and to the suspended mixture were added 0.32 mg (0.0024 mmol) of1-hydroxybenzotriazole and 4.6 mg (0.024 mmol) of1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. Then, theresulting mixture was shaken at a room temperature for 3 days.

The reaction mixture was diluted 100-fold with water, and the dilutedsolution was subjected to a gel-permeation chromatography (AKTA purifiersystem, manufactured by Amarsham Bioscience K.K., column: Superdex 200HR 10/30, flowrate: 0.5 mL/min., eluent: 10 mM phosphate buffer (pH 7.4)containing 150 mM NaCl) to determine the molecular weight of thereaction product. The result confirmed a polypeptide peak in a molecularweight range of 30000 to 200000. The molecular weight was calculatedwith a Gel Filtration LMW Calibration Kit and a Gel Filtration HMWCalibration Kit (manufactured by Amarsham Bioscience K.K.) as referencematerials.

The resulting reaction mixture was diluted 5-fold with water, and thediluted solution was dialyzed against water for 3 days to remove thereagent(s) such as the condensing agent and the unreacted monomer(s).The circular dichroism spectrum was measured in the obtainedpolypeptide, and the result showed that positive Cotton effect wasobserved at a wavelength of 228 nm and negative Cotton effect at awavelength of 198 nm, and confirmed that the polypeptide formed a triplehelical structure.

Example 3

A peptide represented by the formula H—(Pro-Hyp-Gly)₁₀—OH (Sequence IDNo. 3) (manufactured by Peptide Institute, Inc.) was dissolved in wateror water containing 50% of methyl alcohol to prepare a solution having aconcentration of 0.25 mg/mL, and a circular dichroism spectrum thereofwas measured with a spectrometer (J-725, manufactured by JASCO Corp.,light path: 1 mm). In water, the result showed that positive Cottoneffect was observed at a wavelength of 225 nm and negative Cotton effectat a wavelength of 195 nm, and confirmed that the peptide formed atriple helical structure. Moreover, the same solution was subjected to agel-permeation chromatography (AKTA purifier system, manufactured byAmarsham Bioscience K.K., column: Superdex Peptide PE 7.5/300, flowrate:0.25 mL/min., eluent: 10 mM phosphate buffer (pH 7.4) containing 150 mMNaCl) thereby confirming one peak showing a molecular weight of about9000 corresponding to a triple helical structure. The molecular weightwas calculated with a Gel Filtration LMW Calibration Kit (manufacturedby Amarsham Bioscience K.K.), human insulin (manufactured by SigmaCorporation) and glycine (manufactured by Wako Pure Chemical Industries,Ltd.) as reference materials. The proportion of the peptide forming thetriple helical structure was about 100% on the basis of the peak area.Also, in the case of water containing 50% of methyl alcohol, almost thesame results were given, and the proportion of the peptide forming thetriple helical structure was about 100%.

Five (5) mg (0.0016 mmol) of the peptide H—(Pro-Hyp-Gly)₁₀—OH wassuspended in 2 mL dimethyl sulfoxide, and the mixture was stirred at aroom temperature. To the mixture were added 0.23 mg (0.0018 mmol) ofdiisopropylethylamine, 0.24 mg (0.0018 mmol) of 1-hydroxybenzotriazole,and 0.65 mg (0.0034 mmol) of1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, and theresultant mixture was further stirred for 7 days at a room temperature.

The reaction mixture was diluted 20-fold with water, and the dilutedsolution was subjected to a gel-permeation chromatography (AKTA purifiersystem, manufactured by Amarsham Bioscience K.K., column: Superdex 200HR 10/30, flow rate: 0.5 mL/min., eluent: 10 mM phosphate buffer (pH7.4) containing 150 mM NaCl). The result confirmed a polypeptide peakwas observed in a molecular weight range of 60000 to not less than200000. The molecular weight was calculated with a Gel Filtration LMWCalibration Kit and a Gel Filtration HMW Calibration Kit (manufacturedby Amarsham Bioscience K.K.) as reference materials.

The resulting reaction mixture was diluted 5-fold with water, and thediluted solution was dialyzed against water for 3 days to remove thereagent(s) such as the condensing agent and the unreacted monomer(s).The circular dichroism spectrum was measured in the obtainedpolypeptide. The result showed that positive Cotton effect was observedat a wavelength of 225 nm and negative Cotton effect at a wavelength of197 nm, and confirmed that the polypeptide formed a triple helicalstructure.

The water suspension containing the resulting polypeptide was cast on asheet made from a fluorine-containing resin (polytetrafluoroethylene),and the cast sheet was air-dried to obtain a cast film. Gold wasdeposited on the film, and the deposited film was observed with the useof a scanning electron microscope. As a result, a fibrous structure wasobserved as shown in FIG. 1.

Example 4

A peptide represented by the formula H—(Pro-Pro-Gly)₅—OH (manufacturedby Peptide Institute, Inc.) was dissolved in water or water containing50% of methyl alcohol to prepare a solution having a concentration of0.25 mg/mL, and a circular dichroism spectrum thereof was measured witha spectrometer (J-725, manufactured by JASCO Corp., light path: 1 mm).The results in both solutions showed that no positive Cotton effect wasobserved at a wavelength in range of 220 to 230 nm and only negativeCotton effect was observed at a wavelength in range of 200 to 202 nm,and confirmed that the peptide did not form a triple helical structure.Moreover, the same solutions were subjected to a gel-permeationchromatography (AKTA purifier system, manufactured by AmarshamBioscience K.K., column: Superdex Peptide PE 7.5/300, flow rate: 0.25mL/min., eluent: 10 mM phosphate buffer (pH 7.4) containing 150 mMNaCl). The result confirmed one peak showing a molecular weight of notmore than 2000 corresponding to a monomer. The molecular weight wascalculated with a Gel Filtration LMW Calibration Kit (manufactured byAmarsham Bioscience K.K.), human insulin (manufactured by SigmaCorporation) and glycine (manufactured by Wako Pure Chemical Industries,Ltd.) as reference materials. The proportion of the peptide forming thetriple helical structure was 0% on the basis of the peak area.

The peptide H—(Pro-Pro-Gly)₅—OH (3.5 mg (0.0026 mmol)) and the peptideH—(Val-Pro-Gly-Val-Gly)₂—OH (Sequence ID No. 4) (0.92 mg (0.0011 mmol))synthesized in the same manner as in Example 2 were suspended in 1.5 mLdimethyl sulfoxide in a predetermined proportion, and the suspension wasstirred at a room temperature. To the mixture were added 0.52 mg (0.0040mmol) of diisopropylethylamine, 0.51 mg (0.0038 mmol) of1-hydroxybenzotriazole, and 1.45 mg (0.0076 mmol) of1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, and theresultant mixture was further stirred for 7 days at a room temperature.

The reaction mixture was diluted 20-fold with water, and the dilutedsolution was subjected to a gel-permeation chromatography (AKTA purifiersystem, manufactured by Amarsham Bioscience K.K., column: Superdex 200HR 10/30, flow rate: 0.5 mL/min., eluent: 10 mM phosphate buffer (pH7.4) containing 150 mM NaCl) to determine the molecular weight of theresulting polypeptide. The result confirmed a polypeptide peak in amolecular weight range of 80000 to 450000. The molecular weight wascalculated with a Gel Filtration LMW Calibration Kit and a GelFiltration HMW Calibration Kit (manufactured by Amarsham BioscienceK.K.) as reference materials.

The resulting reaction mixture was diluted 5-fold with water, and thediluted solution was dialyzed against water for 3 days to remove thereagent(s) such as the condensing agent and the unreacted monomer(s).The circular dichroism spectrum was measured in the obtainedpolypeptide. The result showed that positive Cotton effect was observedat a wavelength of 227 nm and negative Cotton effect at a wavelength of198 nm, and confirmed that the polypeptide formed a triple helicalstructure.

The water suspension containing the resulting polypeptide was cast on asheet made of a fluorine-containing resin (polytetrafluoroethylene), andthe cast sheet was air-dried to obtain a cast film. The film wasimmerged in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl togive a gel sheet. The gel sheet was transparent at a room temperature,and was reversibly clouded at a temperature of not lower than 40° C.

Example 5

A peptide represented by the formula H—(Pro-Hyp-Gly)₅—OH (Sequence IDNo. 5) (manufactured by Peptide Institute, Inc.) was dissolved in wateror water containing 50% of methyl alcohol to prepare a solution having aconcentration of 0.25 mg/mL, and a circular dichroism spectrum thereofwas measured with a spectrometer (J-725, manufactured by JASCO Corp.,light path: 1 mm). In water, the result showed that positive Cottoneffect was observed at a wavelength of 223 nm and negative Cotton effectat a wavelength of 201 nm, and confirmed that the peptide formed atriple helical structure. Moreover, the same solution was subjected to agel-permeation chromatography (AKTA purifier system, manufactured byAmarsham Bioscience K.K., column: Superdex Peptide PE 7.5/300, flowrate:0.25 mL/min., eluent: 10 mM phosphate buffer (pH 7.4) containing 150 mMNaCl) thereby confirming one peak showing a molecular weight of about4100 corresponding to a triple helical structure. The molecular weightwas calculated with a Gel Filtration LMW Calibration Kit (manufacturedby Amarsham Bioscience K.K.), human insulin (manufactured by SigmaCorporation) and glycine (manufactured by Wako Pure Chemical Industries,Ltd.) as reference materials. The proportion of the peptide forming thetriple helical structure was about 100% on the basis of the peak area.Also, in the case of water containing 50% of methyl alcohol, almost thesame results were given, and the proportion of the peptide forming thetriple helical structure was about 100%.

Five (5) mg (0.0033 mmol) of the peptide H—(Pro-Hyp-Gly)₅—OH wassuspended in 2 mL dimethyl sulfoxide, and the mixture was stirred at aroom temperature. To the mixture were added 0.44 mg (0.0034 mmol) ofdiisopropylethylamine, 0.46 mg (0.0033 mmol) of 1-hydroxybenzotriazole,and 1.3 mg (0.0068 mmol) of1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, and theresultant mixture was further stirred for 14 days at a room temperature.

The reaction mixture was diluted 20-fold with water, and the dilutedsolution was subjected to a gel-permeation chromatography (AKTA purifiersystem, manufactured by Amarsham Bioscience K.K., column: Superdex 200HR 10/30, flow rate: 0.5 mL/min., eluent: 10 mM phosphate buffer (pH7.4) containing 150 mM NaCl). A polypeptide peak was observed in amolecular weight range of 40000 to not less than 100000. The molecularweight was calculated with a Gel Filtration LMW Calibration Kit and aGel Filtration HMW Calibration Kit (manufactured by Amarsham BioscienceK.K.) as reference materials.

The resulting reaction mixture was diluted 5-fold with water, and thediluted solution was dialyzed against water for 3 days to remove thereagent(s) such as the condensing agent and the unreacted monomer(s).The circular dichroism spectrum was measured in the obtainedpolypeptide. The result showed that positive Cotton effect was observedat a wavelength of 224 nm and negative Cotton effect at a wavelength of199 nm, and confirmed that the polypeptide formed a triple helicalstructure.

Example 6

Five (5) mg (0.0016 mmol) of a peptide represented by the formula:H—(Pro-Hyp-Gly)₁₀—OH (Sequence ID No. 3) (manufactured by PeptideInstitute, Inc.) was dissolved in 0.5 mL of 10 mM phosphate buffer(containing 8.1 mM Na₂HPO₄.12H₂O, 1.5 mM KH₂PO₄ and 2.7 mM KCl, pH 7.4),and the solution was stirred at 20° C. To the solution were added 0.24mg (0.0018 mmol) of 1-hydroxybenzotriazole and 31 mg (0.16 mmol) of1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, and theresultant mixture was further stirred for 24 hours at 20° C.

The reaction mixture was diluted 60-fold with water, and the dilutedsolution was subjected to a gel-permeation chromatography (AKTA purifiersystem, manufactured by Amarsham Bioscience K.K., column: Superose 6 HR10/30, flow rate: 0.5 mL/min., eluent: 10 mM phosphate buffer (pH 7.4)containing 150 mM NaCl) to determine the molecular weight of thereaction product. The result confirmed a polypeptide peak correspondingto an average molecular weight of 400000. The molecular weight wascalculated with a Gel Filtration LMW Calibration Kit and a GelFiltration HMW Calibration Kit (manufactured by Amarsham BioscienceK.K.) as reference materials.

Moreover, the resulting reaction mixture was diluted 5-fold with water,and the diluted solution was dialyzed against water for 3 days to removethe reagent(s) such as the condensing agent and the unreactedmonomer(s). The circular dichroism spectrum was measured in the obtainedpolypeptide, and the result showed that positive Cotton effect wasobserved at a wavelength of 225 nm and negative Cotton effect at awavelength of 197 nm, and confirmed that the polypeptide formed a triplehelical structure.

Example 7

A peptide represented by the formula: H—(Pro-Hyp-Gly)₁—OH (manufacturedby Peptide Institute, Inc.) was dissolved in water to prepare a solutionhaving a concentration of 0.25 mg/mL, and a circular dichroism spectrumthereof was measured with a spectrometer (J-820, manufactured by JASCOCorp., light path: 1 mm). The results showed that positive Cotton effectwas observed at a wavelength of 214 nm and negative Cotton effect at awavelength of 196 nm, and confirmed that the peptide did not form atriple helical structure. Moreover, with respect to the same solution,the molecular weight was measured by gel-permeation chromatography (AKTApurifier system, manufactured by Amarsham Bioscience K.K., column:Superdex Peptide PE 7.5/300, flow rate: 0.25 mL/min., eluent: 10 mMphosphate buffer (pH 7.4) containing 150 mM NaCl). The result confirmedone peak showing a molecular weight of about 250 corresponding to amonomer. The molecular weight was calculated with a Gel Filtration LMWCalibration Kit (manufactured by Amarsham Bioscience K.K.), humaninsulin (manufactured by Sigma Corporation) and glycine (manufactured byWako Pure Chemical Industries, Ltd.) as reference materials. Theproportion of the peptide forming the triple helical structure was 0%.

Twenty-five (25) mg (0.088 mmol) of a peptide represented by theformula: H—(Pro-Hyp-Gly)₁—OH was dissolved in 2.5 mL of 10 mM phosphatebuffer (pH 7.4), and the solution was stirred at 20° C. To the solutionwere added 12.1 mg (0.09 mmol) of 1-hydroxybenzotriazole and 168 mg(0.88 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidehydrochloride, and the resultant mixture was further stirred for 24hours at 20° C.

The reaction mixture was diluted 20-fold with water, and the dilutedsolution was subjected to a gel-permeation chromatography (AKTA purifiersystem, manufactured by Amarsham Bioscience K.K., column: Superdex 200HR 10/30, flow rate: 0.5 mL/min., eluent: 10 mM phosphate buffer (pH7.4) containing 150 mM NaCl) to determine the molecular weight of thereaction product. The result confirmed a polypeptide peak in a molecularweight range of 20000 to 30000. The molecular weight was calculated witha Gel Filtration LMW Calibration Kit and a Gel Filtration HMWCalibration Kit (manufactured by Amarsham Bioscience K.K.) as referencematerials.

The resulting reaction mixture was diluted 5-fold with water, and thediluted solution was dialyzed against water for 3 days to remove thereagent(s) such as the condensing agent and the unreacted monomer(s).The circular dichroism spectrum was measured in the obtainedpolypeptide, and the result showed that positive Cotton effect wasobserved at a wavelength of 225 nm and negative Cotton effect at awavelength of 199 nm, and confirmed that the polypeptide formed a triplehelical structure.

Test Example

A cast film was made from each polypeptide manufactured in Examples 1and 3, and the film was pasteurized with 25 kGy gamma irradiation. About10,000 of mouse normal fibroblast strain NIH3T3 were dispersed on thefilm, and was incubated in an Eagle's Minimum Essential Mediumcontaining 10% bovine fetal serum in the presence of 5% CO₂ at 37° C.for 3 days. The NIH3T3 cell was well adhered to and proliferated on thefilm made from each polypeptide obtained in Examples 1 and 3, and was noabnormality in configuration or others.

1. A polypeptide comprising a peptide unit represented by formula (1)below and optionally one or more units represented by formulae (2) to(3) below:[—(OC—(CH₂)_(m)—CO)_(p)-(Pro-Y-Gly)_(n)-]_(a)  (1);[—(OC—(CH₂)_(m)—CO)_(q)-(Z)_(r)-]_(b)  (2); and[—HN—R—NH—]_(c)  (3), wherein “m” denotes an integer of 1 to 18, “p” and“q” are the same or different, each representing 0 or 1, “Y” representsPro or Hyp, and “n” denotes an integer of 1 to 20; “Z” represents apeptide chain comprising 1 to 10 amino acid residue(s), “r” denotes aninteger of 1 to 20, and “R” represents a linear or branched alkylenegroup; the molar ratio of “a” relative to “b” [a/b] is 100/0 to 30/70;when p=1 and q=0, c=a, when p=0 and q=1, c=b, when p=1 and q=1, c=a+b,and when p=0 and q=0, c=0; and wherein the polypeptide shows a peak ofmolecular weight in a range from 60 kD to 1000 kD in the molecularweight distribution in terms of globular protein by means of an aqueousgel permeation chromatography, and at least part of the polypeptideforms a triple helix.
 2. The polypeptide according to claim 1, wherein“m” denotes an integer of 2to 12; “n” denotes an integer of 2 to 15; “Z”is a peptide chain comprising at least an amino acid residue or apeptide residue selected from the group consisting of Gly, Sar, Ser,Glu, Asp, Lys, His, Ala, Val, Leu, Arg, Pro, Tyr, and lie; “r” denotesan integer of 1 to 10; and “R” represents a C₂₋₁₂ alkylene group.
 3. Thepolypeptide according to claim 1, which shows positive Cotton effect ata wavelength in n range of 220 to 230 nm and negative Cotton effect at awavelength in a range of 195 to 205 nm in a circular dichroism spectrum.4. The polypeptide according to claim 1, wherein the polypeptide shows apeak of molecular weight in a range from 60 kD to 200 kD in themolecular weight distribution in terms of globular protein by means ofan aqueous gel permeation chromatography.
 5. The polypeptide accordingto claim 1, wherein the polypeptide shows a peak of molecular weight ina range from 80 kD to 450 kD in the molecular weight distribution interms of globular protein by means of an aqueous gel permeationchromatography.
 6. A biomaterial or biocompatible material comprisingthe polypeptide according to claim
 1. 7. A cosmetic preparationcomprising the polypeptide according to claim
 1. 8. A food additivecomprising the polypeptide according to claim
 1. 9. A composition foruse in a pharmaceutical composition said, composition comprising thepolypeptide according to claim 1 together with a liquid or solidsubstrate.
 10. A medical material comprising the polypeptide accordingto claim
 1. 11. The medical material according to claim 10, wherein themedical material is a tissue engineering carrier, a regenerative medicaltreating carrier, a tissue binding agent, an antiadhesive material, asurgical operating suture, a hemostatic material or a contact lens. 12.A method comprising applying the polypeptide according to claim 1 to atissue of a subject.
 13. A process for producing the polypeptideaccording to claim 1, which comprises reacting a peptide component (A)represented by the following formula (1a) with a peptide component (B)represented by the following formula (2a) and a compound (C) representedby the following formula (3a), as an optional component:X-(Pro-Y-Gly)_(n)—OH  (1a) wherein “X” represents H or the groupHOOC—(CH₂)_(m)—CO— (“m” denotes an integer of 1 to 18), “Y” representsPro or Hyp, and “n” denotes an integer of 1 to 20,X-(Z)_(r)—OH  (2a) wherein “X” represents H or the groupHOOC—(CH₂)_(m)—CO— (“m” denotes an integer of 1 to 18), “Z” represents apeptide chain comprising 1 to 10 amino acid residue(s), and “r” denotesan integer of 1 to 20,H₂N—R—NH₂  (3a) wherein “R” represents a linear or branched alkylenegroup, wherein the ratio of the peptide component (A) relative to thepeptide component (B) [the former (A)/the latter (B)] is 100/0 to 30/70(mol %); provided that in the case where “X” represents the groupHOOC—(CH₂)_(m)—CO— (“m” has the same meaning as defined above) in theformula (1a) and/or (2a), the amount of the compound (C) issubstantially 1 mol relative to 1 mol of the total molar amount of thepeptide component (A) and/or the peptide component (B); and in the casewhere “X”0 represents H in the formula (1a) and/or (2a), the reaction isconducted without the compound (C).
 14. The process according to claim13, wherein the reaction is carried out in the presence of at least adehydrating and condensing agent.
 15. The process according to claim 14,wherein the dehydrating and condensing agent comprises at least onemember selected from the group consisting of a carbodiimide-seriescondensing agent, a fluorophosphate-series condensing agent, and adiphenylphosphorylazide.
 16. The process according tea claim 14 which isconducted in a water-free solvent system, wherein the proportion of thedehydrating and condensing agent is 0.7 to 5 mol relative to 1 mol ofthe total molar amount of said reaction components (1a), (2a) and (3a).17. The process according to claim 14 which is conducted in awater-containing solvent system, wherein the proportion of thedehydrating and condensing agent is 2 to 500 mol relative to 1 mol ofthe total molar amount of said reaction components (1a), (2a) and (3a).18. The process according to claim 13, wherein the reaction is carriedout in the presence of a dehydrating and condensing agent and acondensing auxiliary.
 19. The process according to claim 18, wherein thecondensing auxiliary comprises at least one member selected from thegroup consisting of an N-hydroxypolycarboxylic imide, anN-hydroxytriazole, a triazine, and ethyl ester of2-hydroxyimino-2-cyanoacetic acid.
 20. The process according to claim18, wherein the amount of the condensing auxiliary is 0.5 to 5 molrelative to the total molar amount of said reaction components (1a),(2a) and (3a).
 21. A polypeptide comprising a peptide unit representedby the following formula:-(Pro-Hyp-Gly)_(n)-, wherein “n” denotes an integer of 1 to 15, and thepolypeptide shows a peak of molecular weight in a range from 60 kD to1000 kD in the molecular weight distribution in terms of globularprotein by means of an aqueous gel permeation chromatography, and atleast part of the polypeptide forms a triple helical structure.